Dermatite atópica (DA) é uma dermatose inflamatória, de caráter crônico e recidivante, com alta prevalência mundial, caracterizada por eczema localizado ou generalizado, xerose cutânea e prurido intenso, mais comum em crianças, porém podendo acometer adultos, muitas vezes prejudicando de forma considerável a qualidade de vida dos pacientes e seus familiares. O diagnóstico de alergia inclui testes in vivo (testes cutâneos de hipersensibilidade imediata ou prick test) e testes in vitro com mensuração de anticorpos IgE no sangue. Está disponível método para detecção de alergia baseado no princípio de diagnóstico por componentes, o ImmunoCAP Immunosorbent Allergen CHIP (ImmunoCAP-ISAC) que marca a transição para o diagnóstico molecular de alergia. No presente estudo, tivemos por objetivos determinar o perfil de resposta IgE a alérgenos purificados, naturais ou recombinantes, em pacientes adultos com DA e comparar o valor do diagnóstico de alergia por componentes com métodos diagnósticos existentes. Foram selecionados 39 pacientes adultos com DA dentre aqueles Atendidos nos Ambulatórios de Alergia e Dermatologia do HCFMRP-USP. A idade dos pacientes variou de 14-66 anos (média ± DP 34,3 ± 2.1 anos), 64% mulheres. O tempo médio de doença foi de 16 anos. SCORAD médio foi de 36,6 (2-90). Média geométrica (MG) de IgE total foi 1,963 kU/L (24-63,000 kU/L). Alérgenos de ácaros foram dominantes, com sensibilização a Der p1 e a Der f1, Der p2 e Der f2 em 82% e 85% dos pacientes, com MG 27,6; 50; 39,2; 45,4 ISU-E respectivamente, seguidos de gato (38%), cachorro (36%) e pólen de gramíneas (36%). IgE para alérgenos de baratas, fungos, pólen de árvores, látex e veneno de insetos foi encontrada em menos de 20% dos pacientes em baixos níveis. Sensibilização para castanhas (33%) e camarão (31%) foram os alérgenos mais prevalentes entre os alimentos, enquanto a reatividade IgE para leite e ovo esteve presente em 10% ou menos dos pacientes, com baixos níveis de anticorpos IgE na maioria dos casos. Apenas 6 pacientes (15,4%) apresentaram IgE para o panalérgeno tropomiosina, e 3/39 (7,7%) foram negativos para todos os 112 componentes de alérgenos testados no ImmunoCAP-ISAC, com níveis de IgE total de 24, 38,7 e 156 kU/L. Concluímos que o perfil de sensibilização IgE entre os pacientes adultos com dermatite atópica difere daquele entre os pacientes com alergiarespiratória, apresentando menos sensibilização para baratas e para o panalérgeno tropomiosina, e difere ainda do perfil presente em crianças com DA, com predomínio de sensibilização IgE a castanhas e camarão, e baixa taxa de sensibilização a alérgenos de leite de vaca e ovo. / Atopic dermatitis (AD) is an inflammatory, chronic, relapsing dermatosis with a high global prevalence characterized by localized or generalized eczema, cutaneous xerosis and intense pruritus, more common in children, but it can affect adults, often causing considerable damage to the quality of life of patients and their families. The diagnosis of allergy includes in vivo tests (skin tests of immediate hypersensitivity or prick test) and in vitro tests with measurement of IgE antibodies in the blood. An allergy detection method based on the principle of component diagnosis is available, ImmunoCAP Immunosorbent Allergen CHIP (ImmunoCAP-ISAC), and marks the transition to the molecular diagnosis of allergy. In the present study, we aimed to determine the IgE response profile to purified, natural or recombinant allergens in adult patients with AD and to compare the value of component allergy diagnosis with existing diagnostic methods. Thirty-nine adult patients with AD were selected from among those attending the Outpatient Clinic of Allergy and Dermatology at HCFMRP-USP. The age of the patients ranged from 14-66 years (mean ± SD 34,3 ± 2.1 years), 64% women. The mean duration of illness was 16 years. SCORAD mean was 36,6 (2- 90). Geometric mean (GM) of total IgE was 1,963 kU / L (24-63,000 kU / L). Mite allergens were dominant, with sensitization to Der p1 and Der f1, Der p2 and Der f2 in 82% and 85% of the patients, with GM 27,6; 50; 39.2; 45.4 ISU-E respectively, followed by cat (38%), dog (36%) and grass pollen (36%). IgE for cockroach, fungus, tree pollen, latex and insect venom allergens was found in less than 20% of patients at low levels. Sensitization to nuts (33%) and shrimp (31%) were the most prevalent allergens among foods, while IgE reactivity to milk and egg was present in 10% or less of the patients, with low levels of IgE antibodies in most cases. Only 6 patients (15.4%) presented IgE for the pan-allergen tropomyosin, and 3/39 (7.7%) were negative for all 112 allergen components tested on ImmunoCAP-ISAC, with total IgE levels of 24; 38,7 and 156 kU / L. We concluded that the IgE sensitization profile among adult patients with atopic dermatitis differs from that among patients with respiratory allergy, presenting less sensitization to cockroaches and to the pan-allergen tropomyosin, andalso differs from the profile present in children with AD, with a predominance of IgE sensitization to nuts and shrimp, and low sensitization rate to cow\'s milk and egg allergens.

Exposição a alérgenos de poeira doméstica é fator de risco para o desenvolvimento de doenças alérgicas. Os objetivos foram estudar a resposta inflamatória pulmonar alérgica induzida pela sensibilização i.n. ou s.c. ao extrato do ácaro Blomia tropicalis (Bt) e avaliar a participação da molécula adaptadora Myd88. Animais C57BL/6 sensibilizados com solução de extrato de Bt adsorvido ao alum exibiram altos níveis de IgE e aumento do número de eosinófilos. Contudo, animais sensibilizados apenas com Bt não apresentaram aumento dos níveis de IgE. Assim, animais C57BL/6 ou MyD88KO foram sensibilizados com uma dose maior do extrato de Bt (i.n. ou s.c.) e desafiados i.n. As sensibilizações produziram aumento do número células no BAL e níveis de IgE. Os parâmetros celulares em animais MyD88KO se mostraram inibidos, contudo, os níveis de IgE foram similares aos dos animais WT. Concluindo, o desenvolvimento de inflamação pulmonar alérgica não requer alum e sensibilização i.n. ou s.c. induz o recrutamento de células, dependente de MyD88 e produção de IgE, independe de MyD88. / Exposure to allergens from house dust is a risk factor for the development of allergic diseases. The objectives were to study the allergic lung inflammatory response induced by sensitization i.n. or s.c. to Blomia tropicalis (Bt) extract and determine the role of MyD88 adapter molecule. C57BL/6 mice sensitized with Bt extract solution adsorbed to alum exhibited high levels of IgE and increased numbers of eosinophils. However, animals sensitized only with Bt did not show increased levels of IgE. Thus, C57BL/6 or MyD88KO mice were sensitized with a higher dose of Bt extract (i.n. or s.c.) and challenged i.n. The sensitization produced increased cell number in BAL and IgE serum levels. Cell parameters were inhibited in MyD88KO mice, however, IgE levels were similar to those of WT mice. In conclusion, the development of allergic lung inflammation does not require alum, i.n. or s.c. sensitization induces cell recruitment dependent of MyD88 adaptor molecule and IgE production is independent of MyD88.

Orientador: Gloria Maria Braga Poterio, Ilka de Fatima S. Ferreira Boin / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T04:48:43Z (GMT). No. of bitstreams: 1 Santos_ReginaMariadaSilvaFeu_M.pdf: 2522967 bytes, checksum: 7189a57f4af010fc0ae8d017adcf0e5d (MD5) Previous issue date: 2008 / Resumo: A elaboração da Sistematização da Assistência de Enfermagem é um dos meios de que o enfermeiro dispõe para aplicar seus conhecimentos técnico-científicos e humanos na assistência ao paciente e caracterizar sua prática profissional, colaborando na definição do seu papel. O objetivo do presente estudo, foi contribuir para diminuir o risco de acidentes alérgicos, durante o período perioperatório, quando do atendimento a pacientes alérgicos ao látex, identificando antecedentes de risco e transmitindo-lhes informações sobre a alergia ao látex. Trata-se de um estudo prospectivo, de caráter descritivo e exploratório realizado com pacientes cirúrgicos eletivos, com história pregressa de reação comprovada a produtos que continham látex. Como resultados, foram identificados produtos que continham látex e produtos isentos de látex, para atendimento perioperatório. Por meio da visita pré-operatória, foram identificados fatores que pudessem expor o paciente a riscos. A proposta da confecção de um protocolo perioperatório para atendimento de pacientes alérgicos ao látex foi particularmente importante e encontra-se em fase de pré-teste. É importante destacar que é função do enfermeiro do centro cirúrgico proporcionar estrutura física, recursos humanos e materiais para que o ato anestésico-cirúrgico seja realizado em condições ideais, visando à assistência integral ao paciente, principalmente utilizando dados previamente coletados por meio da Vpré-OE. Dos resultados obtidos nas condições adotadas neste estudo nos permitiram concluir que os fatores de risco detectados, alimentares ou não, coincidem com os citados na literatura, mas diferem quanto à freqüência. Dentre as alergias às frutas, as mais freqüentes, em ordem decrescente, foram maracujá, banana e nozes; e em menor e igual percentual, tomate, abacate, kiwi, abacaxi e laranja. / Abstract: The elaboration of the Systematization of Nursing Assistance is one of the means that the nurse applies his/her technical-scientific and human knowledge on the assistance to the patient and characterizes his professional practice, collaborating on the definition of his/her role. The main objective is to reduce the risk of allergic accidents during the Peroperative period, of attending allergic patients to latex by identifying previous risk patients and providing knowledge concerning latex allergy. It is a prospective descriptive and exploratory study accomplished with surgical chosen patients with former proven history of reaction to products that contained latex. As results, products that contained latex were identified and products exempt from latex for preoperative assistance, as well as by means of a pre-surgical visit, factors that could put the patient at risk were identified. The proposal of making a Peroperative protocol for assisting patients allergic to latex was particularly important and is in phase of pre-testing. It is worth pointing out that due to the CC nurse providing physical structure, human resources and materials for the anesthetic/surgical procedure to be performed in ideal conditions, in view of the complete assistance to the patient, specially using data previously collected by the (Vpré-OE). The results obtained with the conditions adopted in this study allow us to conclude: The risk factors detected, related to nourishment or not, correspond to the ones mentioned in literature, but differ with regards to its frequency. Amongst the allergies to fruits, the most frequent ones in decreasing order were: passion fruit, banana and nuts and in less or similar percentage, tomato, avocado, kiwi, pineapple and orange. / Mestrado / Pesquisa Experimental / Mestre em Cirurgia

Orientador: Ricardo de Lima Zollner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T07:59:14Z (GMT). No. of bitstreams: 1 Ferro_KarlaPriscilaVieira_D.pdf: 2593573 bytes, checksum: dbc61c553ff3b0a43d9d67bfd357abee (MD5) Previous issue date: 2011 / Resumo: As reações alérgicas à ferroada de inseto resultam de resposta exacerbada do sistema imune, com produção de elevados níveis de anticorpos IgE alérgeno-específicos e padrão de citocinas Th2, envolvidas na diferenciação de linfócitos B específicos para aquele antígeno em células produtoras de IgE e recrutamento de células efetoras da resposta alérgica. Neste contexto, granulocitos são células efetoras importantes na fase tardia da resposta alérgica e estão envolvidos na patogênese de diferentes doenças. Eosinófilos e neutrófilos, especificamente, modulam a resposta imune por meio de diferentes mecanismos, como a secreçao de citocinas, quimiocinas e mediadores lipídicos. A IgE desempenha papel central na patogênese das doenças alérgicas, interagindo com dois receptores de membranas: alta afinidade FcsRI e baixa afinidade FcsRII (CD23). A ligação da IgE ao seu receptor em mastocitos e basófilos promove a liberação de mediadores inflamatórios, dentre eles, a histamina. A histamina além de induzir os sintomas agudos da reação alérgica, sustenta a reação inflamatória até a fase crônica, sendo estes efeitos mediados através da ativação de diferentes receptores (H1, H2, H3 e H4). Os fatores liberadores de histamina (HRF), particularmente, HRF-dependentes de IgE, induzem a liberação de histamina na fase tardia da resposta alérgica, permitindo a perpetuação dos eventos inflamatórios crônicos. Muitos estudos demonstram a eficácia da imunoterapia específica na dessensibilização e no desenvolvimento de tolerância em indivíduos com quadros graves de hipersensibilidade à ferroada de insetos, sobretudo da classe Hymenoptera. Com base nestas informações, foram objetivos do presente trabalho avaliar os efeitos modulatórios da imunoterapia sobre a expressão gênica dos receptores de histamina (H1, H2 e H4), HRF- IgE dependente e de fatores apoptóticos (Bcl-2 e BID) por RT-PCR, além da expressão gênica, através da técnica de PCR em tempo real de fatores de transcrição envolvidos na diferenciação de granulocitos como PU.1, C/EBPa, C/EBPpe GATA-1, receptores de alta (FcsRla e FcsRly) e baixa afinidade de IgE (CD23), cuja detecção protéica foi realizada por imunofluorescência e citometria de fluxo, respectivamente. Além disso, foram avaliados os níveis séricos de IgE específica, secreçao de RANTES e IL-8 nos sobrenadantes das culturas celulares e quantificação de granulocitos apoptóticos através da técnica de TÚNEL. Os granulocitos foram isolados de pacientes submetidos à imunoterapia específica ao veneno de abelha, em diferentes períodos do tratamento (Pré, 1, 3, 6, 12, 18 e 24 meses), após injeção subcutânea, e submetidas à cultura por 72 horas, com estimulo de 1 ng/mL veneno de abelha. Indivíduos não alérgicos foram estudados como grupo controle. De maneira geral, a imunoterapia específica ao veneno de abelha foi capaz de modular os elementos analisados, reduzindo significativamente a expressão dos mesmos ao final de 24 meses de tratamento. Não verificamos, apenas, modulação no número de granulocitos apoptóticos ao longo da imunoterapia. Nossos resultados inéditos fornecem informações adicionais sobre os efeitos da imunoterapia sobre granulocitos, reforçando as propriedades supressoras e tolerogênicas desta forma de tratamento / Abstract: Allergic reactions to insect stings results from a exacerbated response of the immune system, resulting in the production of high levels of allergen-specific IgE antibodies and Th2 cytokine pattern, which are involved in the differentiation process of B lymphocytes, specific for that antigen, into IgE producing cells and the recruitment of effector cells of allergic response. Eosinophils and neutrophils, specifically, modulate the immune response through different mechanisms, such as the secretion of cytokines, chemokines and lipid mediators. IgE plays a central role on allergic diseases pathogenesis, interacting with two membrane receptors: high affinity FcsRI and low affinity FcsRII (CD23). Biding of IgE with receptors on mast cells and eosinophils promotes the release of inflammatory mediators, among them, histamine. Histamine, besides inducing acute symptoms of allergic reaction, supports inflammatory response until its chronic stage; these effects are mediated through the activation of distinct receptors (H1, H2, H3 and H4). Histamine releasing factors (HRF), particularly, IgE dependent HRF, induce histamine release during the late phase of allergic response, allowing the perpetuation of chronic inflammatory events. In this context, many studies have demonstrated the efficacy of specific immunotherapy on desensitization and tolerance development in subjects with severe hypersensivity to insect stings, especially Hymenoptera. Based on all these information, the aim of the present study were to evaluate the modulating effects of immunotherapy on gene expression of histamine receptors (H1, H2 and H4), IgE dependent HRF and apoptotic factors (Bcl-2 and BID), through RT-PCR; in addition to gene expression, through real time PCR, transcriptional factors involved at granulocytes differentiation as of PU.1, C/EBPa, C/EBPp and GATA-1, and protein expression of high (FcsRIa e FcsRly)and low affinity (CD23) IgE receptors, assessed by immunofluorescence and flow cytometry, respectively. Serum levels of specific IgE were also assessed, along with RANTES and IL-8 secretion in cell culture supernatant and quantification of apoptotic granulocytes through TUNEL technique. Granulocytes were isolated from patients undergoing bee venom specific immunotherapy in different periods of treatment (Pre, 1, 3, 6, 12, 18 and 24 months), after subcutaneous injection, and cultured for 72 hours, with bee venom 1ng/ml_. Non allergic subjects were studied as control group. Overall, bee venom specific immunotherapy was able to modulate the analyzed elements, significantly reducing their expression at the end of 24 months of treatment. Modulation on the number of apoptotic granulocytes were not observed during immunotherapy. Our results provide additional information about the effects of immunotherapy over granulocytes, reinforcing the suppressor and tolerogenic properties of this treatment / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Orientador: Heloísa Helena de Araújo Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T22:34:56Z (GMT). No. of bitstreams: 1 Benetti_LeticiaRegina_M.pdf: 4223548 bytes, checksum: 318ea90daa8192a20e71e361921955fd (MD5) Previous issue date: 2012 / Resumo: Estudos têm demonstrado que o sulfeto de hidrogênio (H2S) tem um importante papel em vários aspectos da fisiologia e da patologia de diferentes doenças. Este estudo teve como objetivo investigar a ação do H2S nos marcadores de estresse oxidativo e sua interação com o oxido nítrico ('NO) na inflamação alérgica pulmonar de camundongos. Camundongos BALB/c foram sensibilizados com ovalbumina (OVA) e tratados com o doador de H2S, hidrosulfeto de sódio (NaHS), ou com o inibidor da oxido nítrico sinatase induzível (iNOS), 1400W, 30 minutos e 2h antes do desafio com OVA, respectivamente. Vinte e quatro, 48 e 96h após o desafio, os animais foram sacrificados e coletou-se o lavado broncoalveolar para contagem total e diferencial de leucócitos e dosagem dos níveis de NOx; os pulmões foram homogeneizados para análise da expressão das proteínas iNOS, MnSOD e Cu/ZnSOD por Western blotting e verificação dos níveis da 3-nitrotirosina (3-NT) por Slot blotting. A atividade da iNOS foi analisada pela conversão de L-arginina para L-citrulina. Os resultados demonstraram que o tratamento com NaHS inibiu a migração de neutrófilos para os pulmões de camundongos sensibilizados em 24h e 48h e de eosinófilos em 48h após o desafio alergênico, quando comparados com os camundongos controles (não tratados). De maneira semelhante, o tratamento com 1400W inibiu o influxo de eosinófilos para o pulmão dos camundongos alérgicos em 48h. O controles em 48h que foi inibida tanto pelo tratamento com NaHS como pelo 1400W. Neste período,desafio com OVA provocou aumento da expressão da iNOS nos pulmões dos animais observou-se aumento da atividade da NOS Ca -independente nos pulmões dos camundongos controles. O tratamento com 1400W, mas não com NaHS, provocou redução desta atividade. Quando comparados com os animais não desafiados, os níveis de NOx nos controles estavam aumentados em todos períodos estudados. Os tratamentos com 1400W e NaHS reduziram seus níveis em 48h e 96h, respectivamente. A expressão da 3-NT foi reduzida pelo 1400W em 48h, mas nenhum efeito foi observado com o NaHS. O desafio com OVA nos animais controles não alterou a expressão de MnSOD, mas provocou aumento da expressão da Cu/ZnSOD em 48h após o desafio com OVA. Neste período, o NaHS reduziu a expressão da MnSOD e o 1400W a expressão da Cu/ZnSOD. Estes resultados demonstram que o efeito anti-inflamatório do H2S na asma ao inibir a migração de eosinófilos e neutrófilos para os pulmões não é decorrente de mecanismos relacionados à via do 'NO. Se a redução da expressão da MnSOD verificada nos animais tratados com NaHS está relacionada à apoptose de eosinófilos e/ou neutrófilos e, consequentemente, à diminuição do conteúdo destas células no LBA precisa ser verificado / Abstract: Studies have shown that hydrogen sulfide (H2S) has an important role in various physiological and pathological aspects of different diseases. In this study, we verified the influence of H2S on oxidative stress and its interaction the nitric oxide ('NO) pathway in the allergic lung inflammation of mice. BALB/c mice were sensitized with ovalbumin (OVA) and treated the H2S donor, sodium hydrosulfide (NaHS), or with the inducible nitric oxide synthase (iNOS) inhibitor, 1400W, 30 minutes and 2h before OVA-challenge, respectively. Twenty-four, 48 and 96h after challenge, mice were sacrificed and the bronchoalveolar lavage (BAL) was collected to investigate the total and differential leukocyte counts and the NOx leveis. The lungs were homogenized to analyze the iNOS, MnSOD and Cu/ZnSOD protein expressions by Western blotting and the 3-nitrotyrosine (3-NT) leveis by Slot blotting. The iNOS activity was determined by conversion of L-arginine to L-citrulline. Results showed that NaHS-treatment of sensitized mice inhibited neutrophil migration at 24h and 48h, as well as the eosinophil influx to the lungs at 48h after allergen challenge when compared with control mice (untreated). Similarly, 1400W-treatment reduced the content of eosinophils in the BAL of allergic mice at 48 hours. OVA-challenge caused an increase in iNOS expression in the lungs of control mice at 48 hours, which was inhibited by NaHS or 1400W treatments. At this time, an increase in Ca+-independent NOS activity was observed in the lungs of control mice. The 1400W-treatment, but not NaHS-treatment, caused a reduction in this activity. When compared with non-challenged mice, NOx leveis were increased in coritrols at ali times studied. Treatment with 1400W or NaHS reduced their leveis at 48h and 96h, respectively. The 3-NT expression was reduced by 1400W at 48 hours, but no effect was observed with the NaHS-treatment. The OVA-challenge of control mice did not modify the MnSOD expression, but caused increased of Cu/ZnSOD expression at 48 hours. However, the NAHS reduced the MnSOD expression and 1400W inhibited the Cu/ZnSOD expression at this time. These results demonstrated that the anti-inflammatory effect of H2S in asthma by inhibiting eosinophils and neutrophil migration to the lungs is not due to mechanisms associated with 'NO pathway. If the reduced MnSOD expression observed in NaHS-treated mice is related with the eosinophil apoptosis and, consequently, decrease of inflammatory cells content in the BAL needs to be studied / Mestrado / Farmacologia / Mestre em Farmacologia

Orientador: Heloísa Helena de Araújo Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T01:09:24Z (GMT). No. of bitstreams: 1 Campos_Daiana_M.pdf: 5833117 bytes, checksum: 9ef22e0c9d416945c3eddacc3a3bb437 (MD5) Previous issue date: 2012 / Resumo: Diversos estudos mostram que o sulfeto de hidrogênio (H2S) exerce importante papel em diferentes aspectos da fisiologia e da patologia pulmonar. O objetivo deste trabalho foi investigar o efeito do tratamento com um doador de H2S no estresse oxidativo produzido pela inflamação alérgica pulmonar. Camundongos BALB/c foram sensibilizados com ovalbumina (OVA) e tratados com NaHS 30 minutos antes de cada desafio com OVA. Os animais sofreram eutanásia em 24h- 144h após o desafio com OVA, quando foi coletado o lavado bronchoalveolar, para contagem total e diferencial de eosinófilos, e retirados os pulmões. Os pulmões foram homogeneizados para análise da atividade das enzimas aconitase (que possui relação inversa com a produção de O2o- intracelular), fumarase, superóxido dismutase (SOD), catalase, glutationa peroxidase (GPx), glutationa redutase (GR) e para a verificação do conteúdo de substâncias reativas ao ácido tiobarbitúrico (TBARS) por método colorimétrico. Os níveis de glutationa reduzida (GSH) e glutationa oxidada (GSSG) foram determinados por cromatografia. Os resultados dos camundongos não tratados (controle) mostraram que o aumento do conteúdo de eosinófilos para os pulmões ocorreu como uma resposta bifásica entre 48h/96h e 144h após o desafio com OVA. Nestes horários, observamos no grupo controle redução na atividade da aconitase, que foi acompanhada do aumento da peroxidação lipídica (TBARS). A atividade da enzima SOO estava aumentada nestes animais a partir de 24h, seguida de uma redução em 144h, enquanto que as atividades das enzimas catalase e GR estavam aumentadas somente em 48h. Não foram observadas modificações na atividade da GPx, assim como nos níveis de GSH e GSSG, em nenhum dos períodos estudados. O tratamento dos camundongos alérgicos com NaHS reduziu o infiltrado de eosinófilos em 48h e 144h após o desafio antigênico, recuperou a atividade da aconitase e reduziu a peroxidação lipídica, quando comparado aos controles. Nestes horários, o tratamento com o NaHS também aumentou as atividades das enzimas SOD, GPx e GR e os níveis de GSH. Diferente destes resultados verificou-se uma redução da atividade da catalase em 48h, quando comparado ao grupo controle. Em conclusão, nossos resultados sugerem que o H2S exerce efeito benéfico na inflamação alérgica pulmonar ao inibir o recrutamento de eosinófilos para os pulmões. Do mesmo modo, produz um importante efeito no estresse oxidativo ao reduzir a peroxidação lipídica e a produção de O2o- intracelular (determinado pela atividade da aconitase) e ao aumentar a atividade da SOD. As atividades das enzimas GPx e GR também estavam aumentadas contribuindo para o aumento de GSH. Deste modo, o NaHS (e provavelmente outros doadores de H2S) podem apresentar potencialmente importante uso terapêutico para o tratamento de doenças pulmonares caracterizadas pela presença de células inflamatórias e desbalanceamento do equilíbrio oxidante/antioxidante / Abstract: Several studies show that the hydrogen sulfide (H2S) plays an important role in different aspects of lung physiology and pathology. The aim of this study was to investigate a H2S-donor treatment on the oxidative stress produced by pulmonary allergic inflammation. BALB/c mice were sensitized with ovalbumin (OVA) and treated with NaHS, 30 minutes before each OVA-challenge. The animals were sacrificed at 24h-144h after challenge, when bronchoalveolar lavage was collected for total and differential eosinophils counts and the lungs were collected. The lungs were homogenized for aconitase (that has inverse correlation with intracelular O2o- production), fumarase, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR) enzymes analysis and thiobarbituric acid reactive substances (TBARS) measurement by colorimetric methods. The determination of reduced glutathione (GSH) and glutathione disulfide (GSSG) levels were done by chromatography. The results from untreated (control) mice showed that an increase of eosinophil influx to the lungs happened as a biphasic response between 48h/96h and at 144h after OVA-challenge. At the same hours we observed in this group a reduction in aconitase levels that were accompanied by lipid peroxidation (TBARS) increase. The SOD activity was increased in control animals at 24h followed by reduction at 144h, whereas catalase and GR activities were increased only at 48h. There were no changes in GPx activity, neither in GSH nor in GSSG levels at any time- studied. NaHS-treatment reduced eosinophils infiltration at 48h and 144h after antigen challenge when it was observed a recovery in the aconitase activity and a decrease in TBARS lung contents as compared to control mice. It was also observed in these times after antigen challenge increases in SOD, GPx as well as GR activities and in GSH levels. Differing from these results, NaHS-treatment reduced catalase activity at 48h, as compared to control group. In conclusion, our results suggest that NaHS exerts beneficial effect on allergic pulmonary inflammation by inhibiting the eosinophils recruitment to the lungs of mice after OVA-challenge. It also produces a significant effect on oxidative stress by reducing lipid peroxidation and intracelular O2o- production (measured by aconitase activities) as well as by increasing SOD activity. The GPx and GR activities were also improved, and that could account for GSH increase. Therefore, NaHS (and probably others H2S donors) may emerge as potentially useful therapeutic tools for treatment of lung diseases characterized by the presence of inflammatory cells and oxidant/antioxidant imbalance / Mestrado / Farmacologia / Mestra em Farmacologia

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. In particular, they have been implicated in the generation of in vitro cytokine-driven T cell polarization and therefore may determine the vigor, quality and/or longevity of such responses in vivo. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, such as 2,4-dinitrochlorobenzene (DNCB) that result in type 1/type 17 responses in mice, and chemicals that cause sensitization of the respiratory tract and occupational asthma, for example trimellitic anhydride (TMA) that induces preferential type 2 responses in mice. To explore the regulation and maintenance of these divergent responses generated by polarised T cell populations in vivo, BALB/c strain mice were exposed topically DNCB and TMA. DNA from draining lymph nodes (LN) was processed for methylated DNA (5mC) immunoprecipitation (MeDIP) followed by hybridization to a whole-genome DNA promoter array. A higher number of DNCB-associated differently methylated regions (DMR) were identified and there was significant crossover between allergen treatments. Promoter-associated DMR, unique to either DNCB or TMA, were generally hypomethylated. Pathway analyses highlighted a number of immune related pathways, including chemokine and cytokine signalling. A number of these DMR were hypothesised to be candidate biomarkers of chemical allergy. To confirm this, novel analysis of hydroxymethylated (5hmC) DNA in the in vivo allergen-activated LN was compared to analysis of 5mC to identify LN specific DMR. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses and the Nwc DMR was characteristic of TMA treatment, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but also provide a possible basis for differentiation between classes of chemical allergens. Finally, a combined population of effector/effector memory T cells (TEff/TEM) was isolated from the CD4+ and CD8+ populations of allergen-activated draining lymph nodes (LN). Levels of 5mC and 5hmC at T cell lineage cytokine prompters was determined and analysed by comparison with concurrently sorted naïve T cells. In CD8+ TEff/TEM from DNCB-stimulated LN, increased expression of Ifng and Gzmb correlated with a reduction 5mC at their respective promoters. There were also reduced levels of 5mC at an Ifng enhancer. In contrast, TMA-simulated CD4+ TEff/TEM were characterised by high levels of Il4 expression which were associated with a decrease in promoter 5mC and an increase in 5hmC, as well as increased 5hmC at an Il4 enhancer region. These data demonstrate that exposure to chemical allergens results in characteristic DNA methylation patterns indicative of epigenetic regulation of divergent T cell populations in vivo. Furthermore, it highlights a particularly important role for DNA hydroxymethylation at the Th2 locus. In conclusion, exposure to chemical allergens results in divergent patterns of 5mC and 5hmC. These provide possible biomarkers for the different classes of chemical allergens and represent an insight into the importance of 5mC and 5hmC in the control of polarised T cell responses in vivo.

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Increased awareness of protection against infections such as Hepatitis Band Human Immune-deficiency Virus (HIV)/ Acquired Immune Deficiency Syndrome (AIDS) and other sexually transmitted diseases has led to an increase in the demand for latex gloves and condoms leading to an increase in latex allergy. Besides latex, condoms also contain several undisclosed chemicals including antioxidants, accelerators, emulsifiers, stabilizers, lubricants, and in some cases flavourings and colourants. Though extensive testing is done to evaluate the physical quality of condoms, little information is available regarding the biological safety of condoms. In this study a modification of the direct cell culture testing method that is specified by the American Test Method F813-83 of 1998 was used to determine the cytotoxicity of the surface material of latex condoms prepared at time intervals that represents normal physiological exposure times T2, T4 and T8. The L929 cells were exposed to medium containing increasing amounts of condom washings (0-66%) for 20 hours. After exposure cell number and viability was determined using the Crystal violet (CV) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-3H-tetrazolium bromide (MTI) assays respectively. Data was evaluated using a split-plot design with the appropriate Analysis of Variance (ANOVA). The effect of the condom washings on cell morphology and CV staining, MTI metabolism and Neutral red (NR) uptake at a fixed condom washing (16%) and exposure time T8 was evaluated microscopically. Cell membrane integrity was evaluated by Propidium iodide (PI) uptake and with PI staining after fixation and Hoechst 33324 (H33342) staining nuclear structure was evaluated with fluorescence microscopy. Apoptosis induced DNA fragmentation was evaluated by agarose gel electrophoresis. The effects of condom washings at 16% condom washing and exposure times T2, T4 and T8 was further evaluated in the HeLa cell line, a cell line in origin and type closer to that of the cervical lining. Cytotoxicity was evaluated using the CV, MTT and NR assays. In the L929 cell line, condom types Non-lubricated condoms (NLC), Lubricated condoms (LC) and Lubricated and flavoured condoms (LFC) behaved differently over time of exposure and the concentrations of condom washings. LFC were found to induce a decrease in cell number compared to other condom types, followed by LC and NLC revealed increases in cell number. Split-plot analysis, revealed that condom type x time (CT x Time) is significantly different due to the effect observed at T2 for LC. The MTT usually considered being more sensitive than the CV assay showed only toxicity for LFC and not for NLC and LC as with the CV assay. Exposure to LFC revealed significant decrease of 70 % decrease in cell viability at T8. Condom washings, LC, LFC and LFCC had no effect on cell morphology following CV staining. MTT metabolism and NR uptake was reduced and altered cell morphology was observed for L929 cells exposed to LFC and LFCC. Little PI uptake was observed for all cells exposed to condom washings. Condensed nuclei were observed for L929 cells exposed to LFC and LFCC while Hoechst staining revealed peripheral arrangement of DNA with Hoechst 33342 staining. Cell death in L929 cells were found to be mediated by apoptosis with L929 exposed to LFC showing the most damage. All effects of LFC is greater than that observed for LFCC indicating that other factors rather than the number of components present in each type of condom may account for toxicity. Toxicity of condom washings were compared to that found in the L929 cell line using the CV and MTT assays and an additional bioassay the NR assay was included. Condom types, LC, LFC and LFCC had a significant effect on cell viability and lysosomal membrane integrity. Differences observed between the L929 and HeLa cells were due to the increased viability observed for LC and the decrease in membrane integrity for LFC on HeLa cells. With LC and LFC no decrease in cell number and viability was observed as previously reported for the L929 cell line. Although no decreased in cell viability is observed for LFC a decrease of 75% in lysosomal membrane integrity is observed. The increase in cell viability found for HeLa exposed to LC (although statistically not significant) cannot be explained. Changes in cell viability and membrane integrity was only observed for HeLa cells, indicating that the HeLa cell line is more sensitive to the cytotoxic effects of condom washings. Furthermore the NR assay is a more sensitive assay than the MTT assay in detecting the cytotoxic effects of LFC condom washings at low concentrations. These are assays address only the effects of short-term exposure and not possible genotoxic effects that may occur following repeated and long-term exposure as reported in other latex products. / Dissertation (MSc (Anatomy))--University of Pretoria, 2006. / Anatomy / unrestricted

Protostrongylus stilesi, the parenchymal lungworm, has been attributed an important role in the widespread respiratory diseases of the Rocky Mountain bighorn sheep (Ovis canadensis canadensis). This study was conducted to delineate some of the immunologic and non-specific interactions between parasite and host. A procedure was developed for- the immunochemical quantitation of the ovine immunoglobulins IgG(1), IgG(2) and IgM and for the semi-quantitative analysis of IgA. This technique obviated, the preparation of highly specific antisera required for single radial immunodiffusion. The lungworm did not appear to be a significant part of the total antigenic environment of infected animals since no relationship between immunoglobulin levels and parasite activity was detected. Genetic influences were suggested in the levels of IgG(2) which remained at a relatively constant level characteristic of individual animals. The seromucoids were evaluated as correlates of the inflammatory reaction to parasitism. These proteins were useful in detecting changes in parasite activity and bacterial infection. Gastrointestinal disorders associated with severe scouring were accompanied by the disappearance of circulating orosomucoid (α-1 acid glycoprotein). The loss of this low molecular weight protein appeared to be related to vascular leakage. Lungworm infections induced the appearance of homocytotropic antibodies which could be detected in vitro by their ability to sensitize peripheral polymorphonuclear leucocytes for adherence to the larval cuticle. The elution of this antibody from sensitized cells and the inhibition of the adherence reaction with specific antiserum suggested that the reaction was mediated at least partly by IgG(1). The adherence reaction was used to assay homocytotropic activity of serum from infected animals (washed-cell test). This test was correlated with the ability of sera to sensitize skin for anaphylaxis. The effective hypersensitive response, accounting for both sensitizing and blocking activity, was determined by exposing normal cells to larvae in a medium containing serum (decomplemented-serum test). The results of this test paralleled inflammatory changes in parasitized animals, monitored by the levels of serum orosomucoid. Using this method, levels of homocytotropic and blocking antibodies were measured throughout the annual parasite cycle. Preliminary observations indicated that immunogenic inflammation, associated with the "spring rise" and "self cure," resulted from a shift in a dynamic balance between competing antibodies rather than a proportionate increase in homocytotropic activity. The response of peripheral lymphocytes to the presence of larvae or larval extracts was cursorily examined. Although ovine lymphocytes did not respond well enough in culture to draw definitive conclusions, the presence of larval extracts appeared to have a detrimental effect on cell survival and transformation. Even in the presence of EDTA, minute amounts of antibody sensitized lymphocytes for adherence to the larval cuticle. Sera from a wide variety of sources had an inhibitory effect on this reaction. Treatment of larvae with neuraminidase and high concentrations of NaCl engendered improved adherence. This suggested that Protostrongylus had adopted mechanisms similar to the trophoblast or the neoplastic cell which allegedly evade immunologic recognition with a strongly anionic coat. These aspects are discussed in terms of the possible role of Protostrongylus in respiratory diseases of the bighorn sheep, and the exploitation of molecular mechanisms in the control of parasitic diseases. / Land and Food Systems, Faculty of / Graduate